The annual contribution of the aquatic species culture to the total production of fish and shellfish has increased in recent times. However, higher densities imply that these animals live in stress conditions and, in general, they are more susceptible to infections. Disease outbreaks caused by pathogens in aquaculture result in large economic losses and even sometimes produce the removal of certain culture from a specific region.
In salmonids, the infestations caused by the infectious pancreatic necrosis virus (IPNV), the viral hemorrhagic septicemia virus (VHSV), the infectious salmon anemia virus (ISAV) and the infectious hematopoietic necrosis virus (IHNV) are the main causes of disease outbreaks in their culture (Marroqui et al. (2008) Antiviral Research 80: 332-338). For example, it is estimated that Chilean salmon production for the year-end 2010 will fall by 38.7% (from 403 000 tonnes in 2009 until 245 000 tonnes in 2010) as a result of mortality caused by ISAV.
Moreover, the crustaceans are one of the most important economic sectors in global aquaculture, contributing over 10 billion annually. The shrimps in culture are susceptible to a wide variety of pathogens including viruses. It is estimated than in the mid-90 approximately 40% of world production (equivalent to 3 billion U.S. dollars) was lost as a consequence of diseases. Five viruses are the main contributors to these losses: the white spot syndrome virus (WSSV), the yellow head virus (YHV), the taura syndrome virus (TSV), the infectious hematopoietic necrosis virus (IHNNV) and the monodon baculovirus (MBV) (reviewed by Johnson et al. (2008) Vaccine 26: 4885-4992).
Bivalves are an important part of the global shellfish production. These organisms, having the characteristic of being filter feeders, are important virus reservoirs; thus their culture may face epizootics which threat its production. It has been reported mass mortalities of adult's oysters of Crassostrea angulata associated with viral infections similar to iridovirus. In addition, other viruses of Herpesviridae, Papovaviridae, Togaviridae, Retroviridae, Reoviridae, Birnaviridae and Picornaviridae families are capable to infect bivalve's cultures. However, due to the absence of cell lines derived from shellfish and the limiting existing molecular tools for these organisms, bivalve virology is still a primitive science based primarily on morphological studies and few experimental studies (reviewed by T. Renault and B. Novoa (2004) Aquat. Living Resour. 17: 397-409).
Antiviral drugs are chemical compounds used to treat infections caused by viruses. The first experimental antivirals were discovered in the early 60's. These were developed based on the “trial” and “error” methodology. However, after the mid 80's, the scene changed dramatically and in recent years many novel antiviral drugs have been developed and registered, mostly for the treatment of the human immunodeficiency virus (HIV). However, since these compounds are not always effective or well tolerated, there are many aspects to be improved. Additional reasons to refine the design and application of these drugs are the emergence of viral resistance or side effects associated with them (De Clercq (2002) Nature Reviews Drug Discovery 1: 13-25). The antiviral drug design has as main targets viral proteins or host cell proteins. The first strategy leads to compounds more specific and less toxic, but with a narrower spectrum of antiviral activity and a greater chance of developing resistance. The second strategy results in discovering antiviral compounds with a broader spectrum of activity, less likely to develop resistance but higher probability to produce cellular toxicity. The strategy of choice depends on the nature of the virus and the potential targets in the virus or in the host cells (De Clercq (2002) Nature Reviews Drug Discovery 1:13-25).
Ribavirin is a broad-spectrum antiviral compound with activity against a wide range of DNA and RNA viruses. It is a nucleoside analogue, which after intracellular phosphorylation, becomes a competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH), a cellular enzyme involved in the synthesis of guanosine monophosphate (GMP) (Graci y Cameron, (2006) Reviews in Medical Virology 16: 49-63; Parker, (2005) Virus Research 107: 165-171). In addition to the inhibition of this enzyme, there are other three mechanisms which have been proposed to explain the antiviral activity of this compound: by direct inhibition of viral RNA polymerase (Toltzis et al. (1988) Antimicrobial Agents and Chemotherapy 32: 492-497), the inhibition of the cap addition at the 5′ end of viral messenger RNA (mRNA) (Goswami et al. (1979) Biochemical and Biophysical Research Communications 89: 830-836) and the accumulation of mutations (Graci y Cameron, (2002) Virology 298: 175-180).
Up to now, there are not specific antiviral drugs approved for the treatment of viral diseases in aquatic organisms. In Chile, a subsidiary of Diagnotec SA named Andrómaco, developed a new antiviral compound called VIROTOP for the treatment of viral diseases in fish. This antiviral is pending for approval by the “Servicio Agrícola y Ganadero” (SAG). However, a significant number of compounds have been evaluated in vitro and in vivo (Hudson et al. (1988) Antiviral Research 9:379-385; Jasher et al. (2000) Antiviral Research 45: 9-17; Kamei and Aoki, (2007) Archives of Virology 152: 861-869; LaPatra et al. (1998) Fish and Shellfish Immunology 8: 435-446; Mas et al. (2006) Antiviral Research 72: 107-115; Micol et al. (2005) Antiviral Research 66: 129-136; Moya et al. (2000) Antiviral Research 48: 125-130), showing various degrees of effectiveness.
The administration of a ribavirin analog, 5-ethynyl-1-β-D-ribofuranosylimidazole-carboxamide (EICAR) has been evaluated in vivo in larvae of salmon coho (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss) experimentally infected with IPNV (Moya et al. (2000) Antiviral Research 48: 125-130). Treatment consisted of daily baths in a solution of 0.4 and 0.8 μg/mL of EICAR for two hours during 20 days. The results showed that the survival of the infected groups and treated with EICAR was similar to survival in the groups not infected with the virus. An analysis of viral load in liver and spleen was performed using the technique of reverse transcription chain reaction polymerase (RT-PCR). This analysis demonstrated a decrease in viral load in animals treated with the antiviral. From this study, it was concluded that EICAR is an inhibitor of IPNV replication, so that their use to reduce viral load and prevent fish mortality is a beneficial tool for increasing crops productivity. However, antiviral treatment is not useful for breeding selection, because infected and treated fish still carry the virus and can transmit it to offspring. On the other hand, it has observed that one of the damage caused by some viral infections is the weight loss in infected fish (Wolf (1986) The fish viruses. In: Espinosa de los Monteros, J., Labarta, U. (Eds.), Patologia en Acuicultura. Industrias gráficas, Españ a, SL, pp. 93-95; Moya et al. (2000) Antiviral Research 48: 125-130). An additional advantage of the use of antivirals is that this weight loss is much lower in fish treated. This effect was most evident in trout than in salmo coho (Moya et al. (2000) Antiviral Research 48: 125-130).
Pituitary adenylate cyclase activating polypeptide (PACAP) belongs to the superfamily of secretin/glucagon/vasoactive intestinal peptide. This peptide was first isolated from bovine hypothalamus in 1989. It was demonstrated its ability to stimulate the secretion of growth hormone through activation of adenylate cyclase and subsequent stimulation of the production of adenosine monophosphate (cAMP) (Miyata et al (1989) Biochem Biophys Res Commun 164 567-574). PACAP is a multifunctional neuropeptide that plays important roles as neurotransmitter, neuromodulator and vasodilator in mammals (Arimura A. (1998) Japanese Journal of Physiology 48:301-31). It has been demonstrated its function in cell division regulation, differentiation and cell death (Sherwood et al. (2000) Endocrine Review 21: 619-670). This peptide exists in two different molecular variants: 27 aa (PACAP27) and 38 aa (PACAP38) (Miyata et al. (1990) Biochemical and Biophysical Research Communications 170:643-8). The effects of PACAP are exerted through a family of three VIP/PACAP receptors that belong to the secretin G-protein-coupled receptor. VPAC-1 and VPAC-2 receptors exhibit similar affinities for the two neuropeptides, VIP and PACAP, whereas PACAP receptor (PAC-1) exhibits a higher affinity for PACAP than for VIP (Vaudry et al. (2000) Pharmacol Rev 52: 269-324). PACAP is widely distributed in the mammalian brain, mainly in the hypothalamus, the paraventricular and dorsamedial nuclei of the thalamus, in the septum, the cerebral cortex, the amygdala, the hippocampus and the cerebellum (Montero et al. (2000) Journal of Molecular Endocrinology 25: 157-168). The most abundant variant in the central nervous system and peripheral tissues is PACAP38. The studies performed in mammals showed the presence of PACAP also in gonads (Shioda et al. (1994) Endocrinology 135: 818-825), adrenal glands (Arimura et al. (1991) Endocrinology 129: 2787-2789), parathyroid glands (Vaudry et al. (2000) Pharmacol Rev 2000; 52: 269-324), endocrine pancreas (Arimura y Shioda (1995) Neuroendocrinology 16: 53-88) and the gastrointestinal tract (Arimura et al. (1991) Endocrinology 129: 2787-2789; Vaudry et al. (2000) Pharmacol Rev 52: 269-324; Hannibal et al. (1998) Cell. Tissue. Res. 291: 65-79).
PACAP and its receptors expression in immune cells have been only partially elucidated (Gaytan et al. (1994) Cell Tissue Res 276:223-7; Abad et al. (2002) NeuroImmunoModulation 10:177-86). In mammals, it has been observed constitutive expression of VPAC-1 receptor in peripheral blood lymphocytes in humans and mice lymphocytes and macrophages, whereas the expression of VPAC-2 is inducible in these cells. Moreover, it has been observed constitutive expression of PAC-1 receptor in rat peritoneal macrophages and human myelomonocytic cell line THP-1. Additionally, it has been reported PACAP expression in thymocytes, different subtypes of T cells, B cells, splenocytes and lymph nodes in rats (Delgado et al. (2001) J Biol Chem 276:369-80; Pozo et al. (2003) Trends Mol Med 9:211-7).
In fish, PACAP has been described in the central (especially hypothalamus, brain to and spinal cord) and peripheral nervous system, innervating eyes, pituitary gland, respiratory tract, salivary glands, gastrointestinal tract, reproductive tract, pancreas and urinary tract (Sherwood et al. (2000) Endocrine Review 21: 619-670).
PACAP inhibits the spontaneous apoptosis of thymocytes in rats (Delgado. (1996) Blood 87: 5152-5161). The fact that PACAP controls thymocyte proliferation suggests that this peptide is an important regulator of the maturation of immune cells (Delgado. (1996) Blood 87: 5152-5161).
This peptide has an indirect effect over lymphocytes maturation through the stimulation of interleukin 6 (IL-6) releases by follicular cells in pituitary. The IL-6 stimulates growth and differentiation of B cells and promotes the synthesis and secretion of immunoglobulins by these cells (Tatsuno et al. (1991) Endocrinology 129: 1797-1804; Yada et al. (1993) Peptides 14: 235-239). Additionally, PACAP activates and suppress the inflammatory response through the regulation of IL-6 and IL-10 (Martinez et al. (1996) J Immunol 156(11):4128-36; Martinez et al. (1998) J Neuroimmunol 85(2):155-67); Martinez et al. (1998) J Leukoc Biol 63(5):591-601). In activated macrophages, PACAP inhibit pro-inflammatory cytokines and stimulates the anti-inflammatory cytokines production, allowing the homeostasis of immune system. Besides, PACAP reduces the expression of co-stimulatory molecules B7.1/B7.2 and subsequent activation of T helper cells (Th). On the other hand, PACAP inhibit the production of IL-6 through its receptor PAC-1 in activated macrophages, suppressing inflammation (Martinez et al. (1998) J Neuroimmunol 85(2):155-67; Martinez et al. (1998) J Leukoc Biol. 1998 May; 63(5):591-601). The inhibitory action of PACAP over IL-6 transcription in response to intense inflammatory stimuli or to intoxication helps tissue protection and immune system homeostasis (Martinez et al. (1998) J Neuroimmunol 85(2):155-67; Martinez et al. (1998) J Leukoc Biol. 1998 May; 63(5):591-601). In contrast, PACAP induce the expression of B7.2 and promotes cellular differentiation to Th2 in non-stimulated macrophages (Delgado y Ganea (2001) Arch Immunol Ther Exp (Warsz) 49(2):101-10).
In general, the function of PACAP in modulation of mammal's immune system has been only partially elucidated in recent years. These studies demonstrated that PACAP regulates both, innate and adaptive immune system and modulates pro and anti-inflammatory response. Nevertheless, the existing information about the effect of this peptide on antiviral response is scarce. Recently, it was demonstrated in hepatitis B chronic patients, an increase in plasma PACAP-38 levels once the viremia was eliminated as a consequence of lamivudine treatment (Elefsiniotis et al. (2003) European Journal of Gastroenterology and Hepatology 15: 1209-1216). This finding suggests an effect over T cells immune response which results in a biochemical and histological disease remission in the patient's liver.
In fish, the in vivo studies about PACAP biological function published until now are mainly related with reproduction (Canosa et al. (2008) American Journal of Physiology (Regul Integr Comp Physiol) 295:1815-21), brain development (Sherwood et al. (2007) Peptides 28:1680-7) and appetite (Matsuda et al. (2005) Peptides 26:1611-6; Maruyama et al. (2006) Peptides 27:1820-6). Recently, it was demonstrated the biological function of this neuropeptide in growth and development of different larval teleost species (Lugo et al. (2008) Journal of Endocrinology 197:583-97).
The knowledge about the function of PACAP in the modulation of fish immune response is limited to studies performed by our research group. We demonstrated that recombinant Clarias gariepinus PACAP administration by baths or injection not only promotes growth but stimulates also innate immune parameters (lysozyme, nitric oxide derived metabolites and antioxidant defences) and acquired immunity (IgM) in larvae and juveniles treated (Carpio et al. (2008) Fish and Shellfish Immunology 25:439-45; Lugo et al. (2010) Fish and Shellfish Immunology 29:513-520). These new properties were described in the patent application “Neuropéptidos para el cultivo de organismos acuáticos” (WO2007/059714).
In vertebrates, one of the first defense line against viral infections is the type I interferon (IFN) system. This system is activated by viral induction of type I interferons (IFNα y IFNβ) through the IFN-α/β receptor which triggers signal transduction mediated by JAK-STAT. The induced cytokines produce a cell antiviral state producing the expression of proteins with antiviral activity such as 2′,5′ oligoadenilate synthetase, quinase R and the GTPases Mx (Goodbourn et al. (2000) Journal of General Virology 81: 2341-2364). Recent evidences establish that fish have an IFN system similar to mammals (Robertsen (2006) Fish and Shellfish Immunology 20: 172-191; Robertsen (2008) Fish and Shellfish Immunology 25: 351-357). The interferons have been cloned in several fish species like zebrafish (Danio rerio), American catfish (Ictalurus punctatus) and Atlantic salmon (Salmo salar) (Altmann et al. (2003) Journal of Virology 77: 1992-2002; Lutfalla et al. (2003) BMC Genomics 4: 29; Robertsen et al. (2003) Journal of Interferon and Citokine Research 23: 601-612; Long et al. (2006) Fish and Shellfish Immunology 21: 42-59). Additionally, it has been identified several IFN regulatory factors, molecules of the JAK-STAT signaling pathway, Mx proteins and other genes stimulated by IFN in different fish species. It was reported antiviral activity mediated by Mx proteins in Salmo salar and Paralichtys olivaceus (reviewed by Robertsen (2006) Fish and Shellfish Immunology 20: 172-191).
Despite the fact that it has not been discovered IFN like genes in crustaceans yet, it has been observed a negative regulation of STAT molecules (these are molecules which are activated in the IFN response in vertebrates) in response of WSSV infection. The negative regulation of STAT during a viral infection antagonizes the type I IFN response in mammals (reviewed by Johnson et al. (2008) Vaccine 26: 4885-4992). In bivalves, the knowledge of antiviral response is even less. There are evidences of antiviral activity in the hemolymph of these organisms and it has been suggested the presence of an IFN like mechanism (Olicarda et al. (2005) Antiviral Research 66(2-3): 147-152; Defer et al., (2009) Aquaculture 293(1-2): 1-7).
In aquaculture, it is of great interest the development of new compounds or compositions that can be employed in the control of viral infections, due to the damages that they cause in this activity.